The cystic fibrosis transmembrane conductance regulator gene (CFTR) encodes an integral membrane protein having structural similarity to the STE6 gene product of yeast, a transporter of the mating pheromone a- factor. Sequence identity. Sequence identity occurs primarily in the first nucleotide binding fold (NBD1), where the major mutation (a deletion of phenylalanine 508) that causes CF is located. To determine the extent to which the NBD1 of STE6 and CFTR are functionally interchangeable, we constructed STE6/CFTR hybrid genes in which portions of STE6 NBD1 were replaced by the corresponding region of STE6 NBD1 was replaced by the corresponding region of the CFTR gene. Introduction of the delta508 mutation into the hybrid gene greatly reduced a-factor transport function By selecting revertants of the defective mating phenotype of a yeast strain expressing the deltaF508 mutant transporter, we identified suppressor mutations in NBD1 which restore a-factor transporter function. When introduced into CFTR deltaF508, these same suppressor mutations suppress the C1- defect associated with the deltaF508 mutation. We propose to isolate additional NBD1 mutations which suppress the deltaF508 mating defect. By devising a model system that allows us to reconstruct CF mutations in yeast, we can use yeast genetics to "revert' these mutations and learn how the CF defect can be reversed. In addition to suppressor mutations in NBD1, we will screen for suppressor mutations that occur in regions of the transporter other than NBD1, and also in yeast genes other than the a-factor transporter. We will also construct new STE6/CFTR hybrid genes in which we have interchanged NBD2 and transmembrane domains of STE6 with CFTR. These STE6/CFTR hybrid genes will define regions of CFTR that function in common with other transporters. Lastly, we will combine our genetic approach with a biochemical approach to the study of transporter function, by devising an in vitro transport system that allows us to study the interaction of STE6 with its a-factor substrate.